A Case Report of a Rare 46,XX/47,XXY Mosaicism With Ovotesticular Disorder of Sex Development and a Literature Review

Klinefelter syndrome (KS) is a common chromosomal abnormality in males, usually presenting as a 47,XXY karyotype and often underdiagnosed. Rarely, KS occurs as mosaic 46,XX/47,XXY. At the same time, ovotesticular disorder of sex development (OT-DSD) is also a rare condition in which both ovarian and testicular structures are present in the same individual, often associated with a 46,XX karyotype. The combination of mosaic 46,XX/47,XXY with OT-DSD is scarce. Herein, we report a new case of a six-month-old infant with unilateral OT-DSD and a 46,XX/47,XXY mosaic karyotype who presented with atypical genitalia at birth. On examination, the external genitalia showed asymmetry of the labioscrotal folds, an empty right fold, a 2.5 cm phallic structure, and a perineal urethral meatus. Imaging studies revealed a uterus and a vaginal cavity, as well as an ovotestis on the left side and an ovarian remnant on the right side. An unexpected increase in testosterone level was observed. Cytogenetics analysis confirmed a mosaic karyotype with 54% of 46,XX and 46% 47,XXY cells. Molecular genetic analysis revealed no mutations in the genes involved in gonadal development. These findings are discussed and the clinical characteristics of the reported cases of 46,XX/47,XXY with OT-DSD are summarized. In conclusion, atypical genitalia leads to the early diagnosis of the rare 46,XX/47,XXY mosaicism with OT-DSD. Mosaicism should be considered in all cryptorchidism cases. Persistent Müllerian structures were common, and the nearly male phenotype of the external genitalia led parents to prefer the male sex of rearing.


Introduction
Klinefelter syndrome (KS) is a common chromosomal abnormality in humans, affecting approximately one in 600 live-born males [1].Often underdiagnosed until adulthood due to mild clinical symptoms, KS is defined by various features such as testicular abnormalities, gynecomastia, tall stature, sparse body hair, androgen deficiency, hypergonadotropic hypogonadism, and normal to slightly decreased verbal intelligence [2].While 80% of KS cases have a 47,XXY karyotype, the remaining 20% involve other chromosomal abnormalities or mosaicism [3].A mosaic 46,XX/47,XXY karyotype is very rare.On the other hand, ovotesticular disorder of sex development (OT-DSD) is a rare gonadal development disorder, comprising approximately 3-10% of disorder of sex development (DSD) cases [4].This condition is marked by the simultaneous presence of ovarian and testicular tissues (ovotestis) within the same individual [5].Furthermore, OT-DSD can be classified into three types: lateral (ovary on one side and testis on the other), bilateral (ovotestis on both sides), or unilateral (ovary or testis on one side and ovotestis on the other).
Unlike KS, overt atypical genitalia is observed in up to 90% of OT-DSD cases [6].The phenotypic presentation of OT-DSD is highly variable, ranging from typical male to typical female appearances [5,7].The most commonly observed karyotype in OT-DSD cases is 46,XX, followed by mosaic patterns such as 46,XX/46,XY and 46,XY [7].Cases involving OT-DSD and mosaicism are more complex and extremely rare.
In this report, we present the description of a new case of a six-month-old infant with a rare subset of unilateral OT-DSD associated with a rare variant of mosaic KS, 46,XX/47,XXY.Molecular studies were performed on several genes to uncover potential hidden genetic causes not evident through karyotyping and fluorescent in situ hybridization (FISH) alone.Additionally, we summarized the clinical features and gonadal characteristics of similar published cases.

Case Presentation
The patient was a six-month-old infant, the youngest of three siblings, born to non-consanguineous parents.The mother, aged 35 years, had a history of three unexplored miscarriages.The pregnancy was well monitored and carried to term.Delivery was by cesarean section, with a birth weight of 3300 grams.
The infant was admitted to our clinical department on the third day of life for evaluation of atypical genitalia.Clinical examination revealed a reactive and tonic neonate without signs of dehydration.
Examination of the external genitalia showed an asymmetry of the labioscrotal folds, with a slightly pigmented striated appearance.The right labioscrotal fold was empty, while the left contained a 1 x 0.5 cm testis.There was also a 2.5 cm phallic structure and a perineal urethral meatus (Figure 1).The remainder of the physical examination was apparently normal.

FIGURE 1: Appearance of the external genitalia.
A: Asymmetry of the labioscrotal folds.B: Presence of phallic structure.
Radiological evaluation with pelvic and scrotal ultrasound showed a vertically elongated structure behind the bladder, suggestive of a uterus (27 x 13 mm), and a homogenous, well-defined nodular formation in the left external genitalia resembling a testis (13.6 x 6 mm).
A pelvic MRI revealed a right-sided retrovesical uterus (15 mm) with a 2 mm endometrium, a 7 mm vaginal cavity, and small ovarian remnants (3 mm right, 2 mm left).On the left side, a high inguinal structure compatible with a testis (8 mm) with minimal hydrocele was noted, as well as a hypertrophied clitoris.The urethra was the female type, and no prostate or right testis was observed (Figure 2).Chromosome analysis was performed on peripheral blood lymphocytes using R-HG banding with a resolution of 5-10Mb according to standard procedures.Karyotyping analysis showed mosaicism with two types of cells: a predominant cell line of normal female chromosome 46,XX in 54% of the examined cells (14 cells), and a 47,XXY cell line in 46% of the examined cells (12 cells) (Figure 3).FISH using the probe LSI SRY/CEPX (Vysis, Abbott, Abbott Park, IL) confirmed the presence of SRY and the mosaicism.The parents had normal karyotypes.Sanger sequencing was performed for the SRY gene.Additionally, we investigated the roles of 49 genes (Appendices) involved in sexual differentiation disorders by sequencing specific regions (coding exons) using the Ion GeneStudio™ S5 System (Life Technologies Corporation, Carlsbad, CA).DNA samples were prepared using the Ion AmpliSeq™ Kit for Chef DL8 and the Ion Chef™ System (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol.The DNA libraries were quantified using the Ion Library TaqMan™ Quantitation Kit, then templated on Ion 530™ Chips with the Ion Chef™ System and sequenced on the Ion GeneStudio™ S5.The raw data generated were analyzed using both Torrent Suite (Thermo Fisher Scientific) and Ion Reporter (Thermo Fisher Scientific), which together provide a comprehensive bioinformatics workflow.Torrent Suite was used initially for quality control, including the removal of low-quality reads and trimming of adapter sequences.It also facilitated the alignment of reads to the reference genome and the initial variant calling, identifying single nucleotide variants (SNVs), insertions, deletions (indels), and copy number variations (CNVs).Following this, Ion Reporter was utilized for further variant analysis.This includes detailed annotation, where variants were cross-referenced with multiple databases for information on gene function, pathogenicity, and population frequency.Filtering steps were applied to focus on variants of clinical significance, based on quality metrics and known disease associations.The analysis culminated in the generation of detailed reports summarizing the identified variants, their potential impacts, and clinical interpretations, thus providing valuable insights into the genetic factors contributing to sexual differentiation disorders.Yet, this analysis did not detect any mutations in all related genes.
The patient, initially assigned male, is currently under follow-up by a multidisciplinary team.The parents are open to reconsidering the child's sex based on gonadal function assessment results and future fertility prospects.Surgical interventions and hormonal treatments will be evaluated in light of these factors.

Discussion
Mosaic 46,XX/47,XXY is a rare variant of KS.Unlike the more common non-mosaic form (47,XXY), which is typically caused by paternal non-disjunction of the sex chromosomes during meiosis [19], the origin of the two cell lines in the 46,XX/47,XXY variant is more complex.In our patient's case, the most likely explanation is a post-zygotic loss of a Y chromosome from an initial 47,XXY zygote, rather than the loss of an X chromosome, giving rise to the 46,XY cell line.The possibility of parental gonadal mosaicism is an unlikely cause of our patient's genotype.
The phenotypic heterogeneity and mild clinical signs of KS often lead to late diagnosis until adulthood, resulting in a lack of early intervention and treatment [2].However, the presence of additional signs of another pathology may allow an earlier detection and more rapid diagnosis.This was observed in patients with associated OT-DSD who present with overt atypical genitalia at birth or during childhood [10,12,15,17].Furthermore, OT-DSD is a rare condition and even rarer when associated with 46,XX/47,XXY mosaic.
According to the literature (Table 2), the age of diagnosis for OT-DSD shows two major peaks.The first peak occurs in childhood (4/12 cases), with ages ranging from 27 weeks of gestation to 1.7 years, often due to the presence of atypical genitalia [10,12,15,17].Only one patient was diagnosed later at 16 years old and had atypical genitalia [13].As seen in our patient, atypical genitalia was identified immediately after birth, leading to an early diagnosis.The second peak occurs after 12 years of age (8/12 cases), where complaints commonly include scrotal pain with or without hematuria (four cases), followed by gynecomastia, female body habitus, and a scrotal mass in three cases.All pubertal patients were reared as male due to their male phenotypic appearance or behavior, though a complete male phenotype is rare.Only one case was reported for near-complete male external genitalia in a 14.5-year-old boy evaluated initially for hematuria [14].
While female or near-female external genitalia are observed separately in OT-DSD or 46,XX/47,XXY individuals [20,21], none of the reported cases with OT-DSD and 46,XX/47,XXY mosaicism presented with completely or nearly female external genitalia, thus, none were raised as female.Gynecomastia was present in all patients over the age of 12 years.Including our patient, 83.3% (10/12) of all individuals had unilateral or bilateral cryptorchidism [5,[10][11][12][13][14]17,18].In our patient, a uterus (15 mm) with endometrium and a vaginal cavity were observed.Similar to most reported cases, the presence of Müllerian derivatives was confirmed in all but one case, which only had Wolffian ducts [8].This latter was present in 41.6% (5/12) of cases with a uterus [11,12,15,17,18].The coexistence of Wolffian and Müllerian derivatives in the same patient with an ovary on one side and an ovotestis on the other has not been reported.
Our 46,XX/47,XXY patient is unique as the ovarian structure was located on the right side of the abdomen, while the left side contained an ovotestis.This differs from all previously reported cases of 46,XX/47,XXY with both an ovary and an ovotestis [5,13,16].Moreover, gonads can be found in various positions, including labioscrotal folds, inguinal regions, or the pelvic/abdominal area.When data were available, the ovary was often located in the abdomen rather than the inguinal or labioscrotal folds, while the ovotestis could be found in all positions without a specific pattern (Figure 4).The position of the gonads primarily seems to depend on the amount of testicular tissue present: it is more likely the gonads descend with a greater amount of testicular tissue [7].The presence of the Y chromosome, along with the genes involved in gonadal development, plays a crucial role in normal testicular development, contributing to the regression of Müllerian derivatives through the secretion and action of anti-Müllerian hormone and facilitating the formation of Wolffian derivatives through the local action of testosterone [22,23].In mosaic cases, gonadal differentiation toward either pure testes or pure ovaries depends on the predominant cell line within the gonads [7].Our patient's molecular analysis, which revealed no mutations in the related genes, supports this explanation.Notably, a reported case of sex discordance in monozygotic 46,XX/47,XXY twins revealed a higher percentage of 46,XX cell lines in all examined tissues, including peripheral blood lymphocytes, buccal smears, and urinary sediments, of the phenotypic girl twin compared to the male twin [24].
In our patient's peripheral blood, the karyotype showed a predominance of 46,XX cells (54%) over 47,XXY cells (46%).This predominance of 46,XX cell lines in peripheral blood has been observed in all reported cases here, regardless of the degree of genital appearance or gonadal differentiation (Table 2).Additionally, there was no correlation between the percentage of Y-bearing chromosome cells in this mosaicism and the degree of masculine appearance observed.The origin of the extra X chromosome is rarely determined; only two cases in the literature have described its origin, and both confirmed a paternal origin [17,25].
In our patient, the high levels of AMH, combined with the persistence of Müllerian ducts and the absence of Wolffian ducts, were intriguing.While the slightly elevated AMH and normal FSH may indicate normal Sertoli cell function [26], the persistence of Müllerian structures might suggest inadequate effectiveness in inducing Müllerian duct regression.The slightly elevated LH and T levels indicate that Leydig cells are somewhat activated but insufficient for complete sexual differentiation.Therefore, the persistence of Müllerian ducts alongside the absence of Wolffian ducts suggests that the hormonal signals for masculinization (testosterone) and Müllerian duct regression (AMH) are not functioning, as they should.
A second hormonal measurement at one month of age revealed that the initially high AMH levels at birth had normalized, indicating a possible typical postnatal adjustment of Sertoli cell function.This change might also suggest that the ovary contributed to the initial higher AMH levels or that there were a higher number of primordial follicles.Nevertheless, the persistently slightly elevated LH and T levels suggest ongoing Leydig cell activity and possible hormonal imbalances.Further exploration of gonadal function could provide insights for determining the most effective therapeutic outcomes.This combination of findings highlights the complex nature of OT-DSD, where hormone levels alone do not fully explain the presence of certain developmental features.Overall, in most cases where data were available, testosterone levels were low, FSH was normal in the prepubertal stage and high during puberty, while LH levels were variable (Table 2).
Furthermore, fertility is reduced in male individuals with OT-DSD or KS [7,27].However, spermatogenesis has been observed in only two OT-DSD individuals [7].Whereas, 21 pregnancies occurred in 10 females with OT-DSD, two of these later developed tumors in their ovotestis.However, none of these females had 46,XX/47,XXY mosaicism [7].Interestingly, paternity might be possible through assisted reproductive techniques like intracytoplasmic sperm injection (ICSI) in both mosaic and non-mosaic KS men [28].Nevertheless, there is an elevated incidence of chromosomal aneuploidies in the offspring [29].To date, there have been no reports of successful reproduction in OT-DSD with 46,XX/47,XXY mosaicism.
Overall, compared to other types of DSDs, OT-DSD patients have a lower risk of germ-cell tumors [7,30].None of the 12 cases reported here documented tumors.However, in our patient, vigilance for potential malignant development with age is recommended, requiring a long-term follow-up.Previous literature has documented that individuals with KS (47,XXY) often experience language disorders and reading disabilities [31].However, these issues are less common in individuals with mosaicism 46,XX/47,XXY [31].Only one patient in this review presented learning difficulties [14].

Conclusions
The presence of atypical genitalia represents a challenge in sex assignment and leads to unpredictable clinical outcomes.It is important to recommend a cytogenetic analysis for all patients with cryptorchidism to detect any hidden mosaicism.Consistent observation of persistent Müllerian structures is a significant finding that may guide management decisions for such cases.The external genitalia predominantly presented a nearly male phenotype, thus influencing the parents' preference for raising their children as male.Furthermore, it is essential to consider fertility potential and genital alignment when deciding on the sex of rearing.No gonadal tumors were documented in the reported cases.Finally, a multidisciplinary management approach is needed, involving geneticists, endocrinologists, surgeons, and psychiatrists to ensure comprehensive care and optimal outcomes for these patients.

FIGURE 2 :
FIGURE 2: Magnetic resonance imaging highlighting the presence of the uterus (red arrow), left testis (yellow arrow), and right ovarian remnant (white arrow).

FIGURE 3 :
FIGURE 3: Karyotype of the patient.A: Karyotype showing a 46,XX cell line.B: Karyotype showing a 47,XXY cell line.

TABLE 3 : List of genes involved in sexual differentiation disorders included in the next- generation sequencing panel.
OMIM: Online Mendelian Inheritance in Man.